US11458106B2 - Lipid nanoparticles comprising lipophilic anti-inflammatory agents and methods of use thereof - Google Patents
Lipid nanoparticles comprising lipophilic anti-inflammatory agents and methods of use thereof Download PDFInfo
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- US11458106B2 US11458106B2 US15/589,181 US201715589181A US11458106B2 US 11458106 B2 US11458106 B2 US 11458106B2 US 201715589181 A US201715589181 A US 201715589181A US 11458106 B2 US11458106 B2 US 11458106B2
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- lipid
- rofleponide
- inflammatory
- peg2000
- budesonide
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Definitions
- lipid nanoparticles facilitate targeted, site-specific delivery of drugs to targeted tissues and cells, thus enhancing their bioavailability.
- a primary obstacle in developing such carriers is the immunogenic response associated with key components of these formulations.
- Liposome carriers suitable for polynucleotide delivery often includes cationic lipid components enabling the delivery across the cellular membrane into the cytoplasm of the target cells.
- cationic liposomes are known to activate the immune system, which has been utilized in vaccines (Peer, D., Advanced Drug Delivery Reviews 64:1738-1748 (2012)).
- LNPs are designed to have a zero net charge at physiological pH 7.4, thus reducing, although not completely eliminating the immune response associated with particles being positively charged.
- liposomes may aid delivery of drugs to target tissues and cell, they also activate the immune system leading to an acute hypersensitivity reaction, which increases the risk of anaphylactic shock.
- anti-inflammatory lipid nanoparticles comprising at least one lipophilic anti-inflammatory agent.
- anti-inflammatory lipid nanoparticles comprising a lipid phase and at least one lipophilic anti-inflammatory agent.
- anti-inflammatory lipid nanoparticles further comprising at least one nucleic acid segment.
- compositions comprising a plurality of anti-inflammatory lipid nanoparticles comprising a lipid phase, at least one lipophilic anti-inflammatory agent and a pharmaceutically acceptable carrier, diluent or excipient.
- pharmaceutical compositions comprising a plurality of anti-inflammatory lipid nanoparticles comprising a lipid phase, at least one lipophilic anti-inflammatory agent, and at least one nucleic acid segment, and a pharmaceutically acceptable carrier, diluent or excipient.
- a method of delivering at least one nucleic acid segment to a target cell comprising contacting the cell with a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- a method of delivering at least one nucleic acid segment to a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- Also disclosed is a method of treating a subject suffering from a disease or disorder comprising administering to the subject a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- methods of inhibiting the immune response associated with non-anti-inflammatory LNP administration comprising administering to a subject a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles disclosed herein.
- the methods of inhibiting the immune response includes inhibiting the expression or activity of at least one biomarker or symptom of an immune response, for instance: (a) inhibiting the expression or activity of pro-inflammatory markers; (b) reducing inflammation (e.g., edema); and (c) reducing production of plasma haptoglobin associated with inflammation or an immune response.
- a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles disclosed herein and a therapeutically effective amount of at least one nucleic acid segment
- processes are disclosed for the preparation of the anti-inflammatory lipid nanoparticles.
- a process for the preparation of anti-inflammatory lipid nanoparticles comprising:
- FIG. 1A illustrates the chemical structures of ionizable lipid components present in the anti-inflammatory lipid nanoparticles
- FIG. 1B illustrates the chemical structures of rofleponide and representative rofleponide prodrugs together with calculated octanol-water log (partition coefficient) using ACD Chemsketch 2014
- FIG. 1C illustrates the chemical structures of budesonide, and representative budesonide prodrugs together with calculated octanol-water log (partition coefficient) using ACD Chemsketch 2014.
- FIGS. 2A-D illustrate (A): edema scoring at 24 hours post administration; (B): plasma haptoglobin concentration at 24 hours post administration; (C): plasma cytokine/chemokine concentrations at 8 and 24 hours post administration; and (D) mRNA 1 protein concentrations vs time after subcutaneous administration of PBS (phosphate buffer, pH 7.4), DLin-MC3-DMA based LNPs without and with rofleponide palmitate (R-C16).
- PBS phosphate buffer, pH 7.4
- R-C16 rofleponide palmitate
- FIGS. 3A-C illustrate (A): edema scoring at 24 hours post administration; (B): plasma haptoglobin concentration at 24 hours post administration; (C): mRNA1 protein concentrations vs time after subcutaneous administration of PBS (phosphate buffer, pH 7.4), DLin-MC3-DMA based LNPs without and with rofleponide palmitate (R-C16)/mRNA 1:1 w/w, 1:10 w/w, and 1:30 w/w.
- PBS phosphate buffer, pH 7.4
- R-C16 DLin-MC3-DMA based LNPs without and with rofleponide palmitate
- FIGS. 4A-C illustrate (A): edema scoring at 24, 48 and 72 hours post administration; (B): plasma haptoglobin concentration vs. time; (C): mRNA 1 protein concentrations vs time after subcutaneous administration of LNPs containing rofleponide palmitate (R-C16) or rofleponide.
- FIG. 5 illustrates rofleponide plasma concentration vs time after subcutaneous administration of LNPs containing rofleponide palmitate (R-C16, figure A) or rofleponide (figure B).
- FIGS. 6A-C illustrate (A): edema scoring; (B): plasma haptoglobin concentration at 24 hours after administration; (C): mRNA 1 protein concentrations vs time after subcutaneous administration of LNPs containing rofleponide pro-drugs with different fatty acid chain lengths: rofleponide valerate (C5), rofleponide myristate (C14), rofleponide palmitate (C16), and rofleponide stearate (C18).
- FIGS. 7A-C illustrate (A): edema scoring at 24 hours after administration; (B) plasma haptoglobin concentration at 24 hours after administration; (C) mRNA 1 protein concentrations vs time after subcutaneous administration of DLin-MC3-DMA and Merck-32 LNPs vs. DLin-MC3/rofleponide palmitate (R-C16), and Merck-32/rofleponide palmitate (R-C16)LNPs.
- FIGS. 8A-C illustrate (A): edema scoring at 24 hours after administration comparing different dosages of mRNA 1; (B) plasma haptoglobin concentration at 24 hours after administration; (C) mRNA 1 protein concentrations vs time after subcutaneous administration of DLin-MC3-DMA and Merck-32 (with rofleponide myristate, R-C14) LNPs comparing different doses of mRNA 1.
- FIGS. 9A-C illustrate (A): edema scoring at 24 hours after administration comparing different dosages of mRNA 1; (B) plasma haptoglobin concentration at 24 hours after administration; (C) mRNA 1 protein concentrations vs time after subcutaneous administration of DLin-MC3-DMA, KL10 (without/with R-C14) and Acuitas-5 (without/with R-C14) LNPs comparing different dosages of mRNA 1.
- FIGS. 10A-G illustrate (A): edema scoring; (B): plasma haptoglobin concentration at 24 hours after administration; (C): plasma IL-6 concentrations at 7 and 24 hours post administration; (D): plasma KC concentrations at 7 and 24 hours post administration; (E) plasma IP-10 concentrations at 7 and 24 hours post administration; (F): plasma MCP-1 concentrations at 7 and 24 hours post administration; (G) mRNA 1 protein concentrations vs time after subcutaneous administration of LNPs containing budesonide pro-drugs with different fatty acid chain lengths: budesonide myristate (C14), budesonide palmitate (C16) or budesonide oleate (C18:1).
- the anti-inflammatory lipid nanoparticles have an electron dense nanostructured core produced by microfluidic mixing of lipid-containing solutions in ethanol with aqueous solutions containing nucleic acid segment(s). It is to be understood that the anti-inflammatory lipid nanoparticles disclosed herein do not have continuous aqueous regions exceeding 50% by volume and thus, exclude conventional liposomes such as unilamellar vesicles and the like.
- the anti-inflammatory lipid nanoparticles comprise a lipid phase and at least one lipophilic anti-inflammatory agent.
- the lipid nanoparticles may further comprise at least one nucleic acid segment.
- One aspect of the present disclosure relates to anti-inflammatory lipid nanoparticles having an average particle size of about 200 nm in diameter or less, for example, less than or equal to about 100 nm, or, for instance, less than or equal to about 75 nm.
- the anti-inflammatory lipid nanoparticles have an average particle size ranging from about 50 nm to about 75 nm, for example, about 60 nm to about 65 nm, such as about 64 nm.
- the anti-inflammatory lipid nanoparticles have an encapsulation efficiency (% EE) of nucleic acid segments of about 80% or higher, such as higher than about 90%, such as ranging from about 95%-100%, for example, about 99%.
- encapsulation efficiency refers to the ratio of encapsulated nucleic acid segment in the anti-inflammatory lipid nanoparticles to total nucleic acid segment content in the pharmaceutical composition measured by lysis of the lipid nanoparticles using a detergent, e.g., Triton X-100. (See e.g., Leung et al. (2012) supra).
- the lipid phase of the anti-inflammatory lipid nanoparticles disclosed herein may be constructed from any materials used in conventional nanoparticle technology, for example, ionizable lipids, neutral lipids, sterols, and polymer-conjugated lipids so long as the net charge of the nanoparticles is about zero.
- Non-limiting examples of ionizable lipids include, for instance, lipids containing a positive charge at physiological pH, for example 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, (see e.g., U.S. Pat. No.
- DLin-DMA 1,2-dilinoleyloxy-3-dimethylaminopropane
- DLin-MC3-DMA dilinoleylmethyl-4-dimethylaminobutyrate
- the ionizable lipids may be present in an amount ranging from about 5% to about 90%, such as from about 10% to about 80%, for instance from about 25% to about 75%, for example, from about 40% to about 60%, such as about 50%, molar percent, relative to the total lipid present in the anti-inflammatory lipid nanoparticles.
- Neutral lipids have a zero net charge at physiological pH.
- Non-limiting examples of neutral lipids include those lipids that exist in an uncharged form or neutral zwitterionic form at physiological pH, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and the like.
- DSPC distearoyl phosphatidylcholine
- DOPE dioleoyl phosphatidylethanolamine
- DPPC dipalmitoyl phosphatidylcholine
- DMPC dimyristoyl phosphatidylcholine
- the neutral lipids may be present in an amount ranging from about 1% to about 50%, such as from about 5% to about 20%, for example, 7.5% to about 12.5%, for instance, about 10%, molar percent, relative to the total lipid present in the anti-inflammatory lipid nanoparticles.
- Non-limiting examples of sterols include cholesterol, and the like.
- the sterols may be present in an amount ranging from about 10% to about 90%, such as from about 20% to about 50%, for instance, from about 35%-45%, such as about 38.5%, molar percent, relative to the total lipid present in the anti-inflammatory lipid nanoparticles.
- Polymer-conjugated lipids comprise a lipid portion and a polymer portion, such as pegylated lipids comprising both a lipid portion and a polyethylene glycol portion.
- Non-limiting examples include dimyristoyl phosphatidyl ethanolamine-poly(ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG2000-c-DOPG, and the like.
- the molecular weight of the poly(ethylene glycol) that may be used may range from about 500 and about 10; 000 Da, or from about 1,000 to about 5,000 Da.
- the polymer-conjugated lipids may be present in an amount ranging from about 0% to about 20%, for example about 0.5% to about 5%, such as about 1% to about 2%, for instance, about 1.5%, molar percent, relative to the total lipid present in the anti-inflammatory lipid nanoparticles.
- the anti-inflammatory lipid nanoparticles may be prepared by combining multiple lipid components.
- the anti-inflammatory lipid nanoparticles may be prepared combining an ionizable lipid, a sterol, a neutral lipid, and a polymer-conjugated lipid at a molar ratio of 50:40-x PEG lipid :10:X PEG lipid , with respect to the total lipids present.
- the anti-inflammatory lipid nanoparticles may be prepared combining an ionizable lipid, a sterol, a neutral lipid, and a polymer-conjugated lipid at a molar ratio of 50:37:10:3 (mol/mol), or, for instance, a molar ratio of 50:38.5:10:1.5 (mol/mol), or, for example, 50:39.5:10:0.5 (mol/mol), or 50:39.75:10:0.25 (mol/mol).
- a lipid nanoparticle may be prepared using an ionizable lipid (such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5), a sterol (such as cholesterol), a neutral lipid (such as DSPC), and a polymer conjugated lipid (such as DMPE-PEG2000) at a molar ratio of about 50:38.5:10:1.5 (mol/mol), with respect to the total lipids present.
- an ionizable lipid such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5
- a sterol such as cholesterol
- a neutral lipid such as DSPC
- a polymer conjugated lipid such as DMPE-PEG2000
- an anti-inflammatory lipid nanoparticle comprising an ionizable lipid (such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5), a sterol (such as cholesterol), a neutral lipid (such as DSPC), and a polymer conjugated lipid (such as DMPE-PEG2000) at a molar ratio of about 47.7:36.8:12.5:3 (mol/mol), with respect to the total lipids present.
- an ionizable lipid such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5
- a sterol such as cholesterol
- a neutral lipid such as DSPC
- a polymer conjugated lipid such as DMPE-PEG2000
- an anti-inflammatory lipid nanoparticle comprising an ionizable lipid (such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5), a sterol (such as cholesterol), a neutral lipid (such as DSPC), and a polymer conjugated lipid (such as DMPE-PEG2000) at a molar ratio of about 52.4:40.4:6.4:0.8 (mol/mol), with respect to the total lipids present.
- an ionizable lipid such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5
- a sterol such as cholesterol
- a neutral lipid such as DSPC
- a polymer conjugated lipid such as DMPE-PEG2000
- a non-limiting example is an anti-inflammatory lipid nanoparticle comprising an ionizable lipid (such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5), a sterol (such as cholesterol), a neutral lipid (such as DSPC), and a polymer conjugated lipid (such as DMPE-PEG2000) at a molar ratio of about 53.5:41.2:4.6:0.7 (mol/mol), with respect to the total lipids present.
- an ionizable lipid such as DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, Merck-32, KL10, or Acuitas-5
- a sterol such as cholesterol
- a neutral lipid such as DSPC
- a polymer conjugated lipid such as DMPE-PEG2000
- an anti-inflammatory lipid nanoparticle comprising an ionizable lipid (such as C12-200), a sterol (such as cholesterol), a neutral lipid (such as DSPC), and a polymer conjugated lipid (such as DMPE-PEG2000) at a molar ratio of about 30:50:19:1 (mol/mol), with respect to the total lipids present.
- an ionizable lipid such as C12-200
- a sterol such as cholesterol
- a neutral lipid such as DSPC
- a polymer conjugated lipid such as DMPE-PEG2000
- the selection of ionizable lipids, neutral lipids, sterols, and/or polymer-conjugated lipids that comprise the anti-inflammatory lipid nanoparticles, as well as the relative molar ratio of such lipids to each other, may be determined by the characteristics of the selected lipid(s), the nature of the intended target cells, and the characteristics of the nucleic acid, such as, for example, mRNA, to be delivered.
- the molar percent of ionizable lipid in the anti-inflammatory lipid nanoparticle may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 70%, relative to the total lipids present.
- the molar percent of neutral lipid in the anti-inflammatory lipid nanoparticle may be greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40%, relative to the total lipids present.
- the molar percent of sterol in the anti-inflammatory lipid nanoparticle may be greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40%, relative to the total lipids present.
- the molar percent of polymer-conjugated lipid in the anti-inflammatory lipid nanoparticle may be greater than about 0.25%, such as greater than about 1%, greater than about 1.5%, greater than about 2%, greater than about 5%, or greater than about 10%, relative to the total lipids present.
- the anti-inflammatory lipid nanoparticles may comprise each of the ionizable lipids, neutral lipids, sterols, and/or polymer-conjugated lipids in any useful orientation desired.
- the core of the nanoparticle may comprise an ionizable lipid and a sterol and one or more layers comprising neutral lipids and/or polymer-conjugated lipids may subsequently surround the core.
- the core of the anti-inflammatory lipid nanoparticle may comprise a core comprising an ionizable lipid (e.g., DLin-MC3-DMA), and a sterol (e.g., cholesterol) in any particular ratio, surrounded by a neutral lipid monolayer (e.g., DSPC) of any particular thickness, further surrounded by an outer polymer-conjugated lipid monolayer of any particular thickness.
- an ionizable lipid e.g., DLin-MC3-DMA
- a sterol e.g., cholesterol
- the lipophilic anti-inflammatory agent and nucleic acid segment may be incorporated into any one of the core or subsequent layers depending upon the nature of the intended target cells, and the characteristics of the nucleic acid, such as, for example, mRNA, to be delivered.
- the core and outer layers may further comprise other components typically incorporated into lipid nanoparticles known in the art.
- the molar percent of ionizable lipids, neutral lipids, sterols, and/or polymer-conjugated lipids that comprise the anti-inflammatory lipid nanoparticles may be selected in order to provide a particular physical parameter of the overall lipid nanoparticle, such as the surface area of one or more of the lipids.
- the molar percent of ionizable lipids, neutral lipids, sterols, and/or polymer-conjugated lipids that comprise the anti-inflammatory lipid nanoparticles may be selected to yield a surface area per neutral lipid, for example, DSPC.
- the molar percent of ionizable lipids, neutral lipids, sterols, and/or polymer-conjugated lipids may be determined to yield a surface area per DSPC of about 1.0 nm 2 to about 2.0 nm 2 , for example about 1.2 nm 2 .
- the anti-inflammatory lipid nanoparticles further comprise a therapeutically effective amount of at least one lipophilic anti-inflammatory agent.
- inflammation refers to a biologic response involving an upregulation of the immune system, which may include an increase in protein activity related to inflammation or an immune response (e.g., pro-inflammatory markers such as chemokines and cytokines, production of plasma haptoglobin) and symptoms of inflammation (e.g., pain, heat, redness and/or edema).
- inflammation is acute. In some embodiments, inflammation is chronic.
- anti-inflammatory agent includes agents that elicit a biological or medical response in a subject that reduce inflammation (either acute or chronic) or downregulate the immune response, for example, by reducing or inhibiting enzyme or protein activity related to inflammation or an immune response (e.g., inhibition of pro-inflammatory markers or reduction in the production of plasma haptoglobin); by ameliorating one or more symptoms of inflammation or an immune response (e.g., pain, redness, heat or edema); or by slowing or delaying of the inflammatory process or the immune response.
- lipophilic anti-inflammatory agent refers to anti-inflammatory agents exhibiting a log P value of about 5.0 or greater.
- log P refers to the determination of the logarithmic base-10 function of the Partition Coefficient, P; wherein P is the relative ratio of the concentration of a compound in an organic phase relative to the concentration of the same compound in an aqueous phase.
- the lipophilic nature of anti-inflammatory agents may be achieved by converting hydrophilic moieties of an anti-inflammatory agent to lipophilic moieties, for example, by converting a carboxyl group, amino group, hydroxyl group, or other hydrophilic group to an alkyl and alkyl esters, acid esters, aryl and aryl esters, heteroaryl esters, amide groups, or other lipophilic group.
- the lipophilic anti-inflammatory agents herein exhibit a log P value of about 5 or above.
- Lipophilic anti-inflammatory agents may be synthesized to increase their log P value by modifying the anti-inflammatory agent to become more lipophilic, as described above.
- the anti-inflammatory agent may be prepared via known techniques such as esterification or alkylation of one or more of any hydrophilic groups present on the parent drug molecule. (See e.g., Waring, M J, Expert Opin. Drug Discov., 5(3): 235-248 (2010).
- terapéuticaally effective amount preceding the at least one lipophilic anti-inflammatory agent refers to the amount of anti-inflammatory agent that inhibits and/or ameliorates any or all of the biomarkers or symptoms of an inflammatory response associated with the administration of non-anti-inflammatory LNPs.
- non-anti-inflammatory LNPs refers to lipid nanoparticles that do not comprise an anti-inflammatory agent.
- Known anti-inflammatory agents include, without limitation, corticosteroids (e.g., rofleponide, budesonide, etc.), and cytokine inhibitors (e.g., JAK1, JAK2, JAK3, TRL1-9, NF- ⁇ b, IRAK-1, IRAK-2, IRAK-4, IRF-3, TBK-1, TRAF-3, p38, IKK ⁇ , etc.), and the like.
- corticosteroids e.g., rofleponide, budesonide, etc.
- cytokine inhibitors e.g., JAK1, JAK2, JAK3, TRL1-9, NF- ⁇ b, IRAK-1, IRAK-2, IRAK-4, IRF-3, TBK-1, TRAF-3, p38, IKK ⁇ , etc.
- the lipophilic anti-inflammatory agent is a rofleponide prodrug.
- rofleponide prodrugs include, but are not limited to rofleponide valerate (C5), rofleponide caproate (C6), rofleponide caprylate (C8), rofleponide caprate (C10), rofleponide laurate (C12), rofleponide myristate (C14), rofleponide palmitate (C16), or rofleponide stearate (C18).
- the lipophilic anti-inflammatory agent is a budesonide prodrug.
- Non-limiting examples include budesonide myristate (C14), budesonide palmitate (C16), budesonide stearate (C18), budesonide oleate (C18:1), and budesonide linoleate (C18:2).
- the lipophilic anti-inflammatory agents may be present in an amount ranging from about 0.001% to about 50%, by weight, relative to the total weight of the anti-inflammatory lipid nanoparticles. In some embodiments, the lipophilic anti-inflammatory agents may be present in an amount ranging from about 0.5% to about 20%, such as about 1% to about 10%, for instance, about 8%, by weight, relative to the total weight of the anti-inflammatory lipid nanoparticles.
- the anti-inflammatory lipid nanoparticles may further comprise a therapeutically effective amount of a nucleic acid segment, which may be associated on the surface of the anti-inflammatory lipid nanoparticles and/or encapsulated within the same anti-inflammatory lipid nanoparticles.
- nucleic acid segment is understood to mean any one or more nucleic acid segments selected from antisense oligonucleotides, DNA, mRNAs, siRNAs, Cas9-guideRNA complex, or combinations thereof.
- the nucleic acid segments herein may be wildtype or modified.
- the anti-inflammatory lipid nanoparticles may comprise a plurality of different nucleic acid segments.
- at least one of the nucleic acid segments, wildtype or modified encodes a polypeptide of interest.
- a therapeutically effective amount preceding the at least one nucleic acid segment refers to an amount of nucleic acid sufficient to modulate protein expression in a target tissue and/or cell type. In some embodiments, a therapeutically effective amount of the at least one nucleic acid segment is an amount sufficient to treat a disease or disorder associated with the protein expressed by the at least one nucleic acid segment.
- the weight ratio of total lipid phase to nucleic acid segment ranges from about 40:1 to about 1:1, such as about 10:1. This corresponds to an approximate molar ratio of ionizable lipid to nucleic acid monomer of about 3:1. In yet another example, the weight ratio of total lipid phase to nucleic acid segment ranges from about 30:1 to about 1:1, such as about 17:1, which corresponds to an approximate molar ratio of ionizable lipid to nucleic acid monomer of about 6:1.
- the relative molar ratio of lipid phase and/or lipid phase components to the nucleic acid monomer may be determined by the nature of the intended target cells and characteristics of nucleic acid segment and thus, are not limited in scope to the above-identified embodiments.
- the anti-inflammatory lipid nanoparticles comprise lipophilic anti-inflammatory agents and nucleic acid segment in a weight ratio range of about 10:1 (lipophilic anti-inflammatory agents to nucleic acid segment) to about 1:100.
- the weight ratio of nucleic acid segment to total lipids present in the lipid nanoparticles ranges from about 2:1 to about 1:50, such as about 1:1 to about 1:10.
- compositions of the present disclosure comprise the anti-inflammatory lipid nanoparticles disclosed herein, a therapeutically effective amount of at least one anti-inflammatory agent and a therapeutically effective amount of at least one nucleic acid segment, and/or one or more pharmaceutically acceptable excipient, carrier or diluent.
- pharmaceutically acceptable excipient, carrier or diluent includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
- the pharmaceutical compositions may further comprise at least one tissue-targeting agent, for example, peptide conjugates such as DSPE-PEG3400-CRPPR or DSPE-CRPPR, and the like.
- compositions may be in a form suitable for parenteral administration.
- the compositions comprising anti-inflammatory lipid nanoparticles disclosed herein may be administered to a subject in need thereof intravenously, intradermally, intramuscularly, subcutaneously, sublingual, intratumorally, intracardiac, by intratracheal instillation, bronchial instillation, and/or inhalation.
- the pharmaceutical compositions may be in the form of a sterile injectable aqueous or suspension, which may be formulated according to known procedures.
- a sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally-acceptable buffer.
- the pharmaceutical composition may be lyophilized resulting in the form of a dry powder, wherein the dry powder can be later reconstituted for administration as needed.
- Dry powder compositions may further comprise bulking agents, for example, sucrose or trehalose.
- compositions can be nebulized by use of inert gases. Nebulized suspensions may be breathed directly from the nebulizing device or the nebulizing device can be attached to face masks tent, or intermittent positive pressure breathing machine. Furthermore, solid dosage forms may also be administered via inhalation using dry-powder inhalers. Suspension or dry powder pharmaceutical compositions can be administered orally or nasally from devices which deliver the pharmaceutical composition in an appropriate manner.
- nucleic acid segment that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated and the particular route of administration.
- routes of administration and dosage regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
- kits comprising a pharmaceutical composition comprising a therapeutically effective amount of at least one anti-inflammatory agent and a therapeutically effective amount of at least one nucleic acid segment, as herein disclosed.
- kits may further comprise various conventional pharmaceutical kit components such as containers comprising pharmaceutically-acceptable adjuvants, diluents or carriers, and additional containers readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
- the present disclosure provides a method for administering pharmaceutical compositions comprising a plurality of anti-inflammatory lipid nanoparticles to a subject in need thereof.
- subject includes warm-blooded mammals, for example, primates, cows, pigs, sheep, dogs, cats, rabbits, rats, and mice.
- subject is a primate, for example, a human.
- subject is in need of treatment (e.g., the subject would benefit biologically or medically from treatment).
- the anti-inflammatory lipid nanoparticles disclosed herein may further serve as platforms for selective delivery of, for example, nucleic acid segments to target cells and tissues, such as antisense oligonucleotides, DNA, mRNAs, siRNAs, Cas9-guideRNA complex.
- nucleic acid segments to target cells and tissues, such as antisense oligonucleotides, DNA, mRNAs, siRNAs, Cas9-guideRNA complex.
- a method of delivering at least one nucleic acid segment to a cell comprising contacting the cell, in vitro or in vivo, with a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- the nucleic acid segment modulates expression, for example, by increasing or decreasing expression, or by upregulating or downregulating expression of the polypeptide.
- Another embodiment provides a method for delivering a therapeutically effective amount of at least one nucleic acid segment to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- compositions comprising a plurality of anti-inflammatory lipid nanoparticles and at least one nucleic acid segment disclosed herein may be used to treat a wide variety of disorders and diseases characterized by underexpression of a polypeptide in a subject, overexpression of a polypeptide in a subject, and/or absence/presence of a polypeptide in a subject. Accordingly, disclosed are methods of treating a subject suffering from a disease or disorder comprising administering to the subject a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles and a therapeutically effective amount of at least one nucleic acid segment.
- the anti-inflammatory lipid nanoparticles may inhibit and/or ameliorate the expression or activity of at least one biomarker or symptom of an immune response.
- the methods disclosed herein reduce and/or inhibit inflammation at the injection site (as determined by edema scoring), reduce and/or inhibit production of plasma haptoglobin, and reduce and/or inhibit the amount of pro-inflammatory markers (e.g., cytokines, chemokines).
- the present disclosure includes methods for inhibiting the expression or activity of pro-inflammatory markers, reducing inflammation (e.g., edema), and for reducing production of plasma haptoglobin, comprising administering a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles comprising a therapeutically effective amount of at least one lipophilic anti-inflammatory agent and a therapeutically effective amount of at least one nucleic acid segment.
- a reduction or inhibition of edema is observed.
- a reduction or inhibition in plasma haptoglobin levels is observed.
- a reduction or inhibition of plasma inflammatory markers in observed.
- the target activity of the encapsulated nucleic acid segment is not inhibited.
- methods for increasing protein expression in cells comprising administering a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles disclosed herein comprising a therapeutically effective amount of at least one anti-inflammatory agent and a therapeutically effective amount of at least one nucleic acid segment.
- Non-limiting examples of pro-inflammatory markers include cytokines and chemokines, for instance, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IP-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-16, IL-17, Exotaxin, FGF-basic, G-CSF, GM-CSF, LIF, MIG, MIP-1, MIP-2, MCP-1, INF- ⁇ , INF ⁇ 2, RANTES, TNF ⁇ , and IL-1 ⁇ .
- cytokines and chemokines for instance, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IP-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-16, IL-17, Exotaxin, FGF-basic, G-CSF, GM-CSF, LIF
- the methods disclosed herein reduce and/or inhibit edema, and reduce and/or inhibit the production of plasma haptoglobin and/or reduce and/or inhibit the production of pro-inflammatory markers associated with non-anti-inflammatory LNP administration by a percentage greater than the control levels.
- control level indicates an untreated sample or subject, or a sample or subject treated with lipid nanoparticles without the disclosed lipophilic anti-inflammatory agent.
- a control level is the level of expression or activity in a control sample in the absence of a lipophilic anti-inflammatory agent.
- the methods disclosed herein reduce and/or inhibit the production of IL-6, IL-8, KC, IP-10, and MCP-1 by a percentage of about 80% or greater, comprising administering a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles to a subject in need thereof, for instance about 85% or greater.
- the methods disclosed herein produce substantially no edema at the injection site.
- substantially no edema is understood to mean no visible swelling and/or redness apparent to the naked eye.
- the methods herein inhibit the production of plasma haptoglobin by a percentage of about 60% or greater comprising administering a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles to a subject in need thereof, for instance about 80% or greater.
- the methods herein comprise administering a pharmaceutical composition comprising a plurality of anti-inflammatory lipid nanoparticles to a subject in need thereof while maintaining normal plasma haptoglobin levels.
- the term “normal plasma haptoglobin levels” includes plasma haptoglobin levels in the range of 3200 ng/mL-65000 ng/m L.
- protein expression may be increased by a factor of about 2 up to 24 hours. In another embodiment, protein expression may be increased by a factor of about 3 up to 72 hours.
- a solution of mRNA 1 in citrate buffer was prepared by mixing mRNA 1 dissolved in MilliQ-water, 100 mM citrate buffer (pH 3) and MilliQ-water to give a solution of 50 mM citrate.
- a lipid solution in ethanol (99.5%) was prepared with four different lipid components: ionizable lipid (DLin-MC3-DMA, Merck-32, KL10 or Acuitas-5); cholesterol (Sigma-Aldrich); DSPC (distearoyl phosphatidyl choline, Avanti Polar Lipids Inc); and DMPE-PEG2000 (dimyristoyl phosphatidyl ethanolamine-poly(ethylene glycol) 2000, NOF Corporation).
- FIG. 1A The chemical structures of the ionizable lipids are shown in FIG. 1A .
- the total concentration of lipids in all experiments was 12.5 mM.
- Lipid nanoparticles containing lipophilic anti-inflammatory agent pro-drugs were prepared by adding pro-drug to the lipidic ethanol solution.
- the chemical structures of rofleponide, and the different pro-drugs abbreviated as R-C5, R-C14.
- R-C16 and R-C18 based on the lengths of the fatty acid chains.
- FIG. 1B The chemical structures of budesonide, and the different pro-drugs abbreviated as B-C14, B-C16 and B-C18:1 based on the lengths of the fatty acid chains, are shown in FIG. 1C .
- the first 0.2-0.35 mL and the last 0.05-0.1 mL of the LNP suspension prepared were discarded while the rest of the volume was collected as the sample fraction. From the sample fraction 25 ⁇ L of sample was injected into 975 ⁇ L 10 mM phosphate buffer pH 7.4 and used to measure the intensity averaged particle size on a Malvern ZetaSizer (ZetaSizer Nano ZS, Malvern Instruments Inc., Westborough, Mass., USA) (“pre-dialysis particle size”) and polydispersity index (PDI). The remaining sample volume was transferred immediately to a Slide-a-lyzer G2 dialysis cassette (10000 MWCO, ThermoFischer Scientific Inc.) and dialyzed over night at 4° C.
- the volume of the PBS buffer was 500-1000 ⁇ the sample fraction volume.
- the sample fraction was then collected and from this volume 25 ⁇ L was injected into 975 ⁇ L 10 mM phosphate buffer, pH 7.4, and the particle size was measured once again (post dialysis particle size) as well as PDI.
- the final mRNA concentration and encapsulation efficiency percentage (% EE) was measured by Quant-it Ribogreen Assay Kit (ThermoFischer Scientific Inc.) using Triton-X100 to disrupt the LNPs.
- mice Female, age ⁇ 12 weeks, Crl:Cd1(ICR), Charles River
- PBS negative control
- mRNA 1 formulated in lipid nanoparticles.
- mice Prior to administration, the mice were lightly anaesthetized with Isoflurane 5% and the injection area was shaved.
- the formulations were then injected subcutaneously (5 ml/kg or 0.3 mg mRNA/kg) in the intrascapular region to different groups of mice. Post-dose blood samples were collected and plasma was prepared by centrifugation.
- Plasma concentrations of haptoglobin were measured by EMD Millipore's MILLIPLEX® MAP Mouse Acute Phase panel 2 kit (Merck KGaA, Darmstadt, Germany). The sample was first diluted 1:20 000 with Assay buffer then, together with standard's and QC's placed in a 96 well plate. A solution containing beads were then added. The beads were magnetic microspheres each of which was coated with a specific antibody. The mixture was incubated over night at 4° C. and the reaction mixture was then incubated with Streptavidin-PE conjungate to complete the reaction on the surface of each microsphere. The plate was read on analyzer Luminex® 100. Each individual microsphere was identified and the result of its bioassay quantified based on fluorescent reporter signals. The concentration was measured using Median Fluorescent Intensity (MFI) data using a 5-parameter logistic curve-fitting method.
- MFI Median Fluorescent Intensity
- Plasma concentrations of murine cytokines/chemokines were measured by EMD Millipore's MILLIPLEX® MAP Mouse Cytokine magnetic bead kit (Merck KGaA, Darmstadt, Germany) for the simultaneous quantification of cytokines; IL-6, KC, MCP-1 and IP-10.
- the samples were first diluted 1:2 with Assay buffer then, together with standard's and QC's placed in a 96 well plate. A solution containing beads were added. The beads are magnetic microspheres each of which is coated with a specific antibody. The mixture was incubated over night at 4° C. and the reaction mixture was then incubated with Streptavidin-PE conjungate to complete the reaction on the surface of each microsphere.
- the plate was read on analyzer Luminex® 100. Each individual microsphere was identified and the result of its bioassay quantified based on fluorescent reporter signals. The concentration was measured using Median Fluorescent Intensity (MFI) data using a 5-parameter logistic curve-fitting method.
- MFI Median Fluorescent Intensity
- mRNA 1 protein was measured as a single analyte with the Milliplex Human Liver Protein Magnetic beads [Merck Millipore, Darmstadt, Germany].
- the assay was performed using the Bioplex Multiplex Suspension Array System, Luminex 100TM] and Bioplex Manager 6.1 software curve fitting software [Bio-Rad Laboratories, Hercules, Calif.]. Briefly, the assay was performed according to a modified protocol based upon the manufacturers' instruction, with additional calibration points to extend the lower range of the standard curve. Sample data was back calculated from the standard curve (5PL curve fitting).
- the concentration of lipophilic anti-inflammatory agent in plasma was determined by protein precipitation followed by liquid chromatography with mass spectrometric detection.
- An 50 ⁇ L plasma sample was precipitated with 180 ⁇ L 0.2% formic acid in acetonitrile containing 10 nmol/L of 5,5-Diethyl-1,3-Diphenyl-2-Iminobarbituric acid as volume marker. After vortex for 3 min and centrifugation (4000 rpm, 4° C., 20 min), the supernatant was taken and analyzed. Analysis of the supernatant was performed on a short reversed-phase HPLC column with rapid gradient elution and MS/MS detection using a triple quadrupole instrument with electrospray ionisation and Multiple Reaction Monitoring (MRM) acquisition.
- MRM Multiple Reaction Monitoring
- Example 1 LNPs Containing Rofleponide Palmitate/mRNA (1:1 w/w)
- R-C16 rofleponide palmitate
- rofleponide palmitate (R-C16) incorporated in the LNPs
- 900 ⁇ L of the samples was ultracentrifuged at 500000 g for 60 min and the bottom fraction of 300 ⁇ L and the original sample (not centrifuged) were analyzed for DLin-MC3-DMA and R-C16 content using a HPLC with signals detected using a PDA-CAD detector.
- the CAD signal was used for DLin-MC3-DMA and, for R-C16, the UV-absorbance maximum at 235 nm was used and the concentrations determined with an external standard curve.
- the analyzed ratio between the two components was compared with the ratio in the original sample.
- the concentration of R-C16 in the different fractions from the LNP samples was compared to the concentration of R-C16 in samples containing nanoparticles of only R-C16 and DMPE-PEG2000 (“R-C16 control”).
- R-C16 control the concentration of R-C16 in samples containing nanoparticles of only R-C16 and DMPE-PEG2000
- the HPLC results for the R-C16 LNPs are shown in Table 1b.
- the ratio between the DLin-MC3-DMA and the R-C16 is similar in all fractions and close to what is seen in the original sample indicating incorporation of rofleponide palmitate (R-C16) in the LNPs.
- Example 2 Non-Anti-Inflammatory LNPs Vs. Rofleponide Palmitate/mRNA (1:1 w/w) LNPs
- R-C16 rofleponide palmitate
- FIGS. 2A-D The results obtained for edema scoring, plasma haptoglobin, plasma cytokines/chemokines and plasma mRNA 1 protein concentrations are shown in FIGS. 2A-D .
- the data for cytokines/chemokines are shown at both 8 and 24 hours.
- the results show that by using LNPs according to the present disclosure, biomarkers or symptoms of an immune response such as inflammation (measured as edema scoring), haptoglobin and cytokines/chemokine in plasma, are significantly reduced.
- the LNPs according to the present disclosure give rise to an increased protein expression.
- Example 3 Non-Anti-Inflammatory LNPs Vs. Rofleponide Palmitate/mRNA (1:1, 1:10 and 1:30 w/w) LNPs
- R-C16 rofleponide palmitate
- the final molar composition of the four lipids and R-C16 in the LNPs are stated in Table 3 below. 1.29 mL of mRNA 1 solution and 0.43 mL of lipid and R-C16 solution was mixed according to the description above.
- the measured pre- and post-dialysis particle sizes as well the % EE are presented in Table 3 below.
- FIGS. 3A-C The results obtained for edema scoring, plasma haptoglobin, and plasma protein concentrations are shown in FIGS. 3A-C .
- the results show that by using R-C16 LNPs (at all ratios), inflammation measured as edema scoring and haptoglobin is significantly reduced.
- the R-C16 LNPs (at all ratios) give rise to an increased protein expression vs. non-anti-inflammatory DLin-MC3-DMA LNPs.
- R-C16 rofleponide palmitate
- Sample S10 was prepared by adding a 16 ⁇ L ethanol solution of 21 mM rofleponide to DLin-MC3-DMA:DSPC:Cholesterol:DMPE-PEG2000 LNPs, after the dialysis step, to give the desired composition and was thereafter equilibrated for 2 days.
- the final molar composition of the four lipids and R-C16 or rofleponide in the LNPs is stated in Table 4 below.
- the measured pre- and post-dialysis particle sizes as well the % EE are presented in Table 4 below (for the size measurement PBS, pH 7.4, was used).
- the results obtained for edema scoring, plasma haptoglobin, and plasma protein concentrations are shown in FIGS. 4A-C .
- the results show that by using R-C16 LNPs according to the present disclosure, inflammation measured as edema scoring and haptoglobin is significantly reduced vs. the LNPs containing the rofleponide at the same molar dose.
- the R-C16 LNPs give rise to an increased and prolonged protein expression protein vs. LNPs containing the rofleponide.
- the results obtained for rofleponide plasma concentration vs. time are shown in FIG. 5 .
- the results show a conversion of rofleponide palmitate to rofleponide after s.c. administration to mice of rofleponide palmitate LNPs.
- the results also clearly illustrate that the rofleponide LNPs gives rise to significantly higher initial plasma concentrations vs when rofleponide palmitate LNPs are used.
- LNPs comprising rofleponide palmitate as compared with LNPs with rofleponide mother compound, where a significant amount of rofleponide leaks into the plasma from the site of administration.
- the LNPs according to the present disclosure result in a total increase of therapeutic window, allowing for significantly smaller doses to be administered to the subject in need.
- Example 6 LNPs with Rofleponide Valerate, Rofleponide Myristate, Rofleponide Palmitate or Rofleponide Stearate
- the final molar composition of the four lipids and R-CX in the LNPs are shown in Table 6 below. 2.475 mL of mRNA 1 solution and 0.825 mL of lipid and R-CX solution was mixed according the general description. The measured pre- and post-dialysis particle sizes as well the % EE are presented in Table 6 below (for the size measurement PBS, pH 7.4, was used).
- the results obtained for edema scoring, plasma haptoglobin, and plasma protein concentrations are shown in FIGS. 6A-C .
- R-C14, R-C16 or R-C18 LNPs give rise to an increased and prolonged protein expression vs. non-anti-inflammatory LNPs, although R-C5 LNPs did not produce a significantly increased total protein expression (0-24 h).
- FIGS. 7A-C The results obtained for edema scoring, plasma haptoglobin, and plasma protein concentrations are shown in FIGS. 7A-C .
- the results show that by using rofleponide palmitate in both DLin-MC3-DMA or Merck-32 based LNPs, inflammation measured as edema scoring and haptoglobin is significantly reduced.
- the presence of rofleponide palmitate in both DLin-MC3-DMA or Merck-32 based LNPs give rise to an increased expression of mRNA 1 protein.
- Example 8 LNPs with Merck-32 with Rofleponide Myristate (at Rolfeponide Myristate/mRNA Ratios 1:1 and 0.3:1 w/w) at a Higher mRNA Dose
- the volume of mRNA 1 solution was 2.13 mL and the volume of the lipid solution was 0.71 mL.
- sample S19 the volume of mRNA 1 solution was 3.12 mL and the volume of the lipid and R-C14 solution was 1.04 mL.
- Sample S20 was composed of two batches identical batches where, for each batch, the volume of the mRNA 1 solution was 3.21 mL and the volume of the lipid and R-C14 solution was 1.07 mL. Both samples S19 and S20 were concentrated using Amicon Ultra-15 Centrifugal Filters to reach a concentration desired for dosing in vivo. Prior to the concentration step, the two batches making up sample S20 were mixed together. The measured pre- and post-dialysis particle sizes as well the % EE are presented in Table 8 below (for the size measurement PBS, pH 7.4, was used).
- the results obtained for edema scoring, plasma haptoglobin and plasma mRNA1 protein concentrations are shown in FIGS. 8A-C .
- the results show that by using rofleponide myristate in Merck-32 based LNPs at a R-C14/mRNA ratio of 1:1, inflammation measured as edema scoring at both 0.3 and 1 mg/kg, is reduced.
- protein expression shows approximate dose proportional increase (over the time interval 0-24 h) when comparing 0.3 and 1 mg/kg doses.
- Example 9 LNPs with KL10 and Acuitas-5 with and without Rofleponide Myristate (at Rolfeponide Myristate/mRNA Ratios 1:1 w/w)
- the molar composition of the lipids used to prepare the lipid solution is stated in Table 9 below.
- the volume of mRNA 1 solution was 2.16 mL and the volume of the lipid solution was 0.72 mL.
- the volume of mRNA 1 protein solution was 1.41 mL and the volume of the lipid and R-C14 solution was 0.47 mL
- the volume of mRNA 1 protein solution was 1.77 mL and the volume of the lipid and R-C14 solution was 0.59 mL.
- the measured pre- and post-dialysis particle sizes as well the % EE are presented in Table 8 below (for the size measurement PBS, pH 7.4, was used).
- FIGS. 9A-C The results obtained for edema scoring, plasma haptoglobin and plasma mRNA 1 protein concentrations are shown in FIGS. 9A-C .
- the results show that by using rofleponide myristate in in both Acuitas-5 and KL10 based LNPs, inflammation measured as edema scoring and plasma haptoglobin is reduced compared to LNPs based on DLin-MC3-DMA (at 0.3 mg/kg).
- the mRNA 1 protein expression (over the time interval 0-24 h) is increased when rolfeponide myristate was incorporated into the LNPs.
- Example 10 LNPs with Merck-32 Alone and with Rofleponide Myristate, Budesonide Myristate, Budesonide Palmitate or Budesonide Oleate
- R-C14 rofleponide myristate
- B-C16 budesonide palmitate
- B-C18:1 budesonide oleate
- the final molar composition of the four lipids and R-C14, B-C14, B-C16 or B-C18:1 in the LNPs are stated in Table 10 below.
- a 3.36 mL aliquot of mRNA 1 protein solution and 1.12 mL of lipid and R-C14, B-C14, B-C16 or B-C18:1 solution was mixed according to the general description described above under the “Preparation of LNPs containing mRNA,” section described above. All the samples were concentrated using Amicon Ultra-4 Centrifugal Filters which were then spun at 3000 rpm and 8° C. until the formulations had an estimated mRNA concentration significantly higher than 0.2 mg/mL.
- the results obtained for edema scoring, plasma haptoglobin, plasma cytokines/chemokines and plasma mRNA 1 concentrations are shown in FIGS. 10A-G .
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JP2022058438A (ja) | 2022-04-12 |
EP3454835A1 (en) | 2019-03-20 |
JP2019514979A (ja) | 2019-06-06 |
JP7066632B2 (ja) | 2022-05-13 |
CN109152735B (zh) | 2022-03-11 |
EP3454835B1 (en) | 2021-03-31 |
US20220370370A1 (en) | 2022-11-24 |
WO2017194454A1 (en) | 2017-11-16 |
US20170367988A1 (en) | 2017-12-28 |
CN109152735A (zh) | 2019-01-04 |
ES2871537T3 (es) | 2021-10-29 |
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